Device and method for detecting the presence of hemoglobin in a biological sample

ABSTRACT

A device and method for detecting the presence of hemoglobin in a biological sample, more particularly, the presence of blood in a fecal sample as an indicator of upper or lower gastrointestinal tract bleeding.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of co-pending prior U.S. application Ser.No. 11/568,382 filed Sep. 11, 2007 which is a national stage entry ofPCT/AU2005/000613 filed Apr. 29, 2005 which claims the benefit of U.S.60/566,731 filed Apr. 30, 2004, all of which are hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to a device and method fordetecting the presence of hemoglobin in a biological sample. Moreparticularly, the present invention provides a device and method fordetecting the presence of blood in a biological sample and still moreparticularly, the presence of blood in a fecal sample as an indicator ofupper or lower gastrointestinal tract bleeding. The method of thepresent invention is useful, inter alia, for the diagnosis ofgastrointestinal tract diseases which can be detected by detectingintestinal bleeding.

BACKGROUND OF THE INVENTION

Bleeding into the bowel is currently the best early indicator of bowelcancer (also know as colorectal cancer). Testing for symptoms ofbleeding into the bowel is usually achieved by screening stools for thepresence of blood. This test is often referred to as fecal occult bloodtesting (referred to as “FOBT”).

Chemical tests are most widely used for FOBT. These tests typicallyrequire stool to be applied to paper impregnated with a chromogenindicator, such as guaiac or 3,3′,5,5′-tetramethylbenzidine (TMB), whichchanges color on oxidation. When developer solution is added to thepaper, a blue color develops with a positive result. Guaiac tests havethe advantage of being inexpensive and easy to perforin, but are lessaccurate (not specific for human blood) and less sensitive thandesirable. Nevertheless, several international studies have shown thatscreening patients with these tests can save lives through the earlydetection of pre-cancerous and cancerous lesions. The commonly usedguaiac tests detect the heme of hemoglobin, and as this is relativelyresistant to breakdown in the small intestine, these tests may detectbleeding anywhere within the intestinal tract. For colorectal cancerscreening this may be a disadvantage as these tumors are confined to thelarge intestine.

Recently, more sensitive and specific immunological tests (e.g.immunochromatographic tests) have been developed that have the potentialto improve the accuracy of detecting blood in screening for colorectalcancer. These tests typically detect the globin protein of hemoglobin, aprotein that does not survive passage through the upper gastrointestinaltract. A positive immunological test therefore indicates lowergastrointestinal bleeding. In common with all immunologically basedtests, however, these tests are subject to a “prozone” or “high dosehook” effect, where at high levels of analyte, the test may be inhibitedto the extent that heavy bleeding may be missed.

Heme from hemoglobin has a pseudoperoxidase activity that catalyses thebreakdown of peroxide substrates and the release of oxygen. The releasedoxygen may be detected by suitable chromogenic indicators such as guaiacand tetramethylbenzidine (TMB) which change color on oxidation. FecalOccult Blood Tests (FOBTs) detect intestinal bleeding by use of thisreaction to detect heme from the hemoglobin of red blood cells, and avariety of formats for such tests are known in the art (see, forexample, U.S. Pat. Nos. 3,996,007; 4,225,557; 4,789,629; 5,064,766;5,100,619; 5,106,582; 5,171,528; 5,171,529 and 5,182,191). Typically,FOBTs involve smearing a stool sample on guaiac-impregnated paper andadding a developer solution containing peroxide. If heme is present, ablue color develops on or around the stool specimen. The disadvantagesof these tests include:

-   -   the stool sample may also contain peroxidases or        pseudoperoxidases from ingested foods and these may cause a        (false) positive reaction in the absence of human blood from the        intestinal tract;    -   heme from ingested meat may also cause a false positive        reaction;    -   the blue color developed with a positive test must be read        against a dark background of stool, so that at lower heme        concentrations the result may be equivocal;    -   with a positive result, color diffuses away from the stool        sample, becomes weaker in intensity, and may fade out (the        transitory nature of the color change may make also make        interpretation of the test result difficult or unreliable);    -   the developer solution, containing peroxide and other reagents,        can interfere with immunochemical tests that may otherwise be        used in conjunction with this test for differentiation between        upper and lower gastrointestinal bleeding (see, for example,        International Patent Publication WO 00/29852, Enterix Inc.,        combining a chromogen test to detect any intestinal bleeding and        an immunochemical test to detect lower intestinal bleeding        only).

FOBTs have also been described that have a peroxide reagent such ascumene hydroperoxide dried in a paper matrix (see, for example, Lam,U.S. Pat. No. 4,071,318). In this case, the test paper can be addeddirectly to water and will develop color if heme is present in thewater. These FOBTs are typically added to a toilet bowl containing astool after a bowel movement in order to detect blood released from thestool into the water. The disadvantages of these tests include:

-   -   blood on, or in, the stool may not diffuse into the water in        sufficient concentration to allow detection;    -   the test must be read against a background of stool and toilet        paper, making interpretation difficult;    -   the tests may also be subject to interference from dietary heme        or peroxidases if there is direct contact between the stool and        test paper;    -   the undeveloped test papers must be stored after manufacture in        desiccated conditions to prevent breakdown of the peroxide        reagent and development of color in the test paper.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

In one aspect, the present invention provides a device for use in thedetection of hemoglobin in a biological sample, particularly a fecalsample, comprising a carrier matrix which includes:

-   (i) a sample application region for receipt of said biological    sample;-   (ii) a substrate region in liquid-conductive communication with, or    combined with, the sample application region and having a    pseudoperoxidase substrate applied thereto or impregnated therein,    said pseudoperoxidase substrate comprising a peroxide or    hydroperoxide reagent; and-   (iii) an indicator region in liquid-conductive communication with,    or combined with, the substrate region and having an indicator    applied thereto or impregnated therein, said indicator producing a    detectable response in the presence of heme and said    pseudoperoxidase substrate.

In one embodiment of this aspect of the invention, the sampleapplication region and the substrate region may be combined into asingle, combined sample application/substrate region having thepseudoperoxidase substrate applied thereto or impregnated therein. Inthe preferred embodiment, however, the sample application region and thesubstrate region are separate regions of the carrier matrix which are inliquid-conductive communication.

In another embodiment, the substrate region and the indicator region maybe combined into a single, combined substrate/indicator region havingboth the pseudoperoxidase substrate and the indicator applied thereto orimpregnated therein. Preferably, however, the substrate region and theindicator region are separate regions of the carrier matrix which are inliquid-conductive communication.

In another aspect, the present invention provides a method for thedetection of hemoglobin in a biological sample, particularly a fecalsample, comprising the steps of:

-   (i) applying said biological sample to a sample application region    of a carrier matrix which comprises said sample application region,    a substrate region and an indicator region;-   (ii) contacting said biological sample with the substrate region    wherein said sample is contacted with a pseudoperoxidase substrate    comprising a peroxidase or hydroperoxidase reagent; and-   (iii) contacting said sample and pseudoperoxidase substrate with the    indicator region wherein said sample and substrate are contacted    with an indicator which produces a detectable response in the    presence of heme and said pseudoperoxidase substrate.

In one embodiment of this aspect of the invention, the biological samplemay be contacted with the pseudoperoxidase substrate in a single,combined sample application/substrate region of the carrier matrix,before permitting or causing flow of the sample and substrate to theindicator region. In a preferred embodiment, however, the sampleapplication region and the substrate region are separate regions of thecarrier matrix which are in liquid-conductive communication, and thebiological sample is applied to the sample application region beforepermitting or causing flow of the sample to the substrate region.

In another embodiment the biological sample may be contacted with thepseudoperoxidase substrate and indicator in a single, combinedsubstrate/indicator region of the carrier matrix, by permitting orcausing flow of the sample from the sample application region to thesubstrate/indicator region. In a preferred embodiment, however, thesubstrate region and the indicator region are separate regions of thecarrier matrix which are in liquid-conductive communication, and thebiological sample is applied to the sample application region beforepermitting or causing flow of the sample to the substrate region, andthen permitting or causing flow of the sample and substrate to theindicator region.

In a particularly preferred embodiment, the device and method of thepresent invention may combine the detection of heme in a biologicalsample as broadly outlined above, with an immunochemical test for thedetection of globin, thereby providing a dual test for differentiationbetween upper and lower gastrointestinal tract bleeding which isparticularly useful for the detection of lower gastrointestinal tractdiseases such as colorectal cancer.

In this preferred embodiment, the carrier matrix of the device asbroadly described above further comprises:

-   (iv) a second substrate region in liquid-conductive communication    with the sample application region and having a detectable    antiglobin immunointeractive molecule applied thereto or impregnated    therein, said immunointeractive molecule forming a detectable    globin-antiglobin complex in the presence of globin; and-   (v) a detection region in liquid-conductive communication with the    second substrate region and having an anti-globin immunointeractive    molecule immobilized therein, said immobilized immunointeractive    molecule immobilizing said detectable globin-antiglobin complex.

Similarly, in this preferred embodiment, the method of the presentinvention as broadly described above further comprises the steps of:

-   (vi) contacting said biological sample with a second substrate    region wherein said sample is contacted with a detectable antiglobin    immunointeractive molecule to form a detectable globin-antiglobin    complex in the presence of globin; and-   (vii) contacting said detectable globin-antiglobin complex with a    detection region wherein said detectable globin-antiglobin complex    is contacted with an immobilized antiglobin immunointeractive    molecule to immobilize said detectable globin-antiglobin complex.

Preferably, the biological sample is permitted or caused to flow fromthe sample application region to the second substrate region which is inliquid-conductive communication with the sample application region, andthe detectable globin-antiglobin complex is permitted or caused to flowfrom the second substrate region to the detection region which is inliquid-conductive communication with the second substrate region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved test format for the detectionof hemoglobin which is particularly suitable for the detection ofhemoglobin as an indicator of intestinal bleeding. The format is alsodesigned to be compatible with immunochemical tests for the detection ofglobin so that a dual test for differentiation between upper and lowerintestinal bleeding is feasible.

The invention involves the use of lateral flow of a liquid sample(suspected of containing blood) from a point of application through oneor more regions containing the reagents required for detection of theheme. The lateral flow layout of the components of the test format hasthe following advantages:

-   -   the color produced with a positive result accumulates as the        flow reaches the end of the carrier matrix, concentrating the        color and facilitating the ease of reading;    -   the color of a positive reaction is free of any fecal or other        obscuring background material;    -   dietary contaminants, such as heme from meat or peroxidases from        food, are diluted on lateral flow, in many cases to below their        threshold detection level;    -   the sample application region may contain enhancing agents that        promote accurate and sensitive detection of the heme by the        downstream test components;    -   incompatible or mutually unstable components or reagents (e.g.        substrate and indicator reagents) may be located in separate        regions, allowing long-term storage without special        manufacturing or storage precautions;    -   as all reagents can be impregnated in the separate regions,        water, water/ethanol mixtures or any other inert reagent may be        used for the test development;    -   the chromogenic heme test (which detects any intestinal        bleeding) may therefore be combined with an immunochemical test        (which is specific for lower intestinal bleeding) so as to allow        for discrimination between upper and lower intestinal bleeding        on the one test sample.

The present invention provides a test device for use in the detection ofhemoglobin in a biological sample, particularly a fecal sample,comprising a carrier matrix which includes:

-   (i) sample application region for receipt of said biological sample;-   (ii) a substrate region in liquid-conductive communication with, or    combined with, the sample application region and having a    pseudoperoxidase substrate applied thereto or impregnated therein,    said pseudoperoxidase substrate comprising a peroxide or    hydroperoxide reagent; and-   (iii) an indicator region in liquid-conductive communication with,    or combined with, the substrate region and having an indicator    applied thereto or impregnated therein, said indicator producing a    detectable response in the presence of heme and said    pseudoperoxidase substrate.    The invention also provides a test method for the detection of    hemoglobin in a biological sample, particularly a fecal sample,    comprising the steps of:-   (i) applying said biological sample to a sample application region    of a carrier matrix which comprises said sample application region,    a substrate region and an indicator region;-   (ii) contacting said biological sample with the substrate region    wherein said sample is contacted with a pseudoperoxidase substrate    comprising a peroxidase or hydroperoxidase reagent; and-   (iii) contacting said sample and pseudoperoxidase substrate with the    indicator region wherein said sample and substrate are contacted    with an indicator which produces a detectable response in the    presence of heme and said pseudoperoxidase substrate.

Reference to a “biological sample” should be understood as a referenceto any sample of biological material derived from an animal such as, butnot limited to, faeces, mucus, urine, biopsy specimens and fluid whichhas been introduced into the body of an animal and subsequently removedsuch as, for example, the saline solution extracted from the lungfollowing lung lavage or the solution retrieved from an enema wash. Thebiological sample which is tested according to the method of the presentinvention may be tested directly or may require some form of treatmentprior to testing. For example, a biopsy sample may requirehomogenisation prior to testing. Further, to the extent that thebiological sample is not in liquid form, (for example it may be a solid,semi-solid or dehydrated liquid sample) it may require the addition of areagent, such as a buffer, to mobilize the sample. The mobilizingreagent may be mixed with the biological sample prior to application ofthe sample to the carrier matrix or the reagent may be applied to thesample after the sample has been applied to the carrier matrix. The useof a mobilizing reagent may also be required to facilitate lateral flow(wicking) of the sample along the carrier matrix. Preferably, thebiological sample is a gastrointestinal sample. By “gastrointestinalsample” is meant any sample which is derived from the gastrointestinaltract. For example, feces, mucus (for example the mucus from a rectalmucus swab), enema wash solution or a gastrointestinal tract biopsysample. Most preferably, the biological sample is a stool sample, or asample of water from a toilet bowl containing a stool.

The term “animal” as used herein includes a human, primate, livestockanimal (e.g. sheep, pig, cow, horse, donkey), laboratory test animal(e.g. mouse, rat, rabbit, guinea pig) companion animal (e.g. dog, cat),captive wild animal (e.g. fox, kangaroo, deer), ayes (e.g. chicken,geese, duck, emu, ostrich), reptile or fish. Preferably, however, theanimal is a human.

Preferably, the carrier matrix used in forming the test device of thepresent invention is in the form of a test strip of a suitable materialwhich permits liquid-conductive communication between the various zonesor regions of the matrix. A particularly preferred material is one whichallows capillary flow, such as an open-celled, chemically inert matrix,with porous plastics material, filter paper and glass fiber beingpreferred. Other suitable materials are well known in the art (see, forexample, Lam U.S. Pat. No. 4,071,318), and are intended to beencompassed within the scope of the present invention.

Preferably, the sample application region of the carrier matrix includesan absorbent pad such as a non-woven polyester pad which is impregnatedwith a reagent to lyse any red blood cells present in the sample so asto release hemoglobin, to minimise binding to the pad and to promotesample flow from the pad. Particularly suitable lysis reagents aredetergents (such as Triton X100). After the sample is applied to thepad, a mobilizing agent such as water or a water/ethanol mixture can beapplied to the pad to mobilize any heme (and globin) in the sample andpermit or cause the sample to flow or wick by capillary action throughthe carrier matrix to the substrate region(s).

The pseudoperoxidase substrate which is present in the substrate regioncomprises a peroxidase or hydroperoxidase reagent as the main reagent,optionally together with supplementary stabilizers, enhancers andaccelerators which are known to persons skilled in the art. Suitableperoxidase or hydroperoxidase reagents include, for example, t-butylhydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide,2,5-dimethylhexane-2,5-dihydroperoxide, paramenthane hydroperoxide ormixtures thereof. Of these, cumene hydroperoxide has been found to bemost preferable. Suitable stabilizing and enhancing agents are also wellknown in the art, and include borate esters such as trimethanolamineborate, triethanolamine borate and tri(n-propanol)amine borate, asstabilizing agents, and 6-methoxyquinoline as an enhancing agent (seeLam U.S. Pat. No. 4,071,318).

The indicator which is present in the indicator region to produce adetectable response in the presence of heme and the pseudoperoxidasesubstrate is preferable a chromogen such as guaiac or a benzidinecompound, for example benzidine, o-tolidine,3,3′,5,5′-tetramethylbenzidine (TMB), 2,7-diaminofluorene, or mixturesof these in varying proportions. Once again, stabilizing agents and/orenhancing agents which are well known to persons skilled in the art maybe included in the indicator.

Preferably, in carrying out the method of the present invention, thesample is applied to an absorbent sample pad in the sample applicationregion where any red blood cells in the sample are lysed by detergent orother lysis reagent in the sample pad to release the hemoglobin. Thesample is then permitted or caused to flow by capillary action from thesample pad to the substrate region using water or water/ethanol as amobilizing agent to develop the test and to solubilize driedpseudoperoxidase substrate (such as cumene peroxide) and otherstabilizing and enhancing reagents located in this substrate region. Thesample and pseudoperoxidase substrate are then permitted or caused toflow by capillary action to the indicator region where the presence ofheme in the sample is detected by a reaction with the chromogen such asguaiac or TMB, resulting in a detectable color change.

In a preferred aspect of the invention, the detection of heme in thebiological sample, as described in detail above, is combined with animmunochemical test in a “dual test” which allows differentiation ofupper and lower intestinal bleeding in the test sample.

Accordingly, in this aspect, the present invention provides a testdevice as broadly described above in which the carrier matrix furthercomprises:

-   (iv) a second substrate region in liquid-conductive communication    with the sample application region and having a detectable    antiglobin immunointeractive molecule applied thereto or impregnated    therein, said immunointeractive molecule forming a detectable    globin-antiglobin complex in the presence of globin; and-   (v) a detection region in liquid-conductive communication with the    second substrate region and having an anti-globin immunointeractive    molecule immobilized therein, said immobilized immunointeractive    molecule immobilizing said detectable globin-antiglobin complex.

The carrier matrix of the test device of the present invention may alsocomprise additional regions to the regions specifically described above.For example, the device may also comprise an absorbent pad or padslocated after the indicator region and/or detection region to draw themobilising liquid front from the sample application region through therespective regions in liquid-conductive communication with each other inorder to develop the tests.

In the preferred aspect, the present invention also provides a method asbroadly outlined above which further comprises the steps of:

-   (vi) contacting said biological sample with a second substrate    region wherein said sample is contacted with a detectable antiglobin    immunointeractive molecule to form a detectable globin-antiglobin    complex in the presence of globin; and-   (vii) contacting said detectable globin-antiglobin complex with a    detection region wherein said detectable globin-antiglobin complex    is contacted with an immobilized antiglobin immunointeractive    molecule to immobilize said detectable globin-antiglobin complex.

Reference throughout this specification to “immunointeractive molecule”should be understood as a reference to any molecule comprising anantigen binding portion or a derivative of said molecule. Examples ofmolecules contemplated by this aspect of the present invention include,but are not limited to, monoclonal and polyclonal antibodies (includingsynthetic antibodies), hybrid antibodies, humanised antibodies,catalytic antibodies) and T cell antigen binding molecules. Preferably,said immunointeractive molecule is an antibody.

Full details of suitable detectable antiglobin immunointeractivemolecules present in the second substrate region, and of suitableimmobilised antiglobin immunointeractive molecules present in thedetection region, are set out in International Patent Publication No. WO00/29852, in the name of Enterix Inc., the contents of which areincorporated herein by reference.

“Detecting” the formation of a globin-antiglobin complex may be by anyconvenient method which will be known to those skilled in the art. Inthe preferred method of the invention described herein, the antiglobinantibody which becomes resuspended by the wicking biological samplefront is complexed with colloidal gold. As theglobin-antiglobin/colloidal gold complex is trapped by the antiglobincapture antibody impregnated in the detection region of the carriermatrix, the colloidal gold becomes visible as a pink band due to itsincreasing concentration during trapping of the complex at this point.Alternatively, the antiglobin antibody may be radio-labeled, orenzymatically labeled such that upon addition of a substrate a colorchange is observed if globin is present.

In one preferred embodiment of the “dual test” aspect of the presentinvention, detection of heme is carried out as described in detailabove. Detection of globin in the biological sample is carried out usinga chromatographic test strip which comprises a second substrate regionand a detection region. The second substrate region is an area ofimmobilized antiglobin antibody coupled to colloidal gold particleswhich are re-suspendible by a passing liquid front, while the detectionregion is an area of immobilized antiglobin capture antibody.

In this preferred aspect of the invention, the biological sample whichis applied to the sample application region flows or wicks to the secondsubstrate region and at this region, the globin component of anyhemoglobin which is present in the sample is bound by the antiglobinantibody coupled to the colloidal gold particles. The passing biologicalsample front re-suspends these antibodies and the globin-antiglobincomplex flows or wicks to the detection region where the globincomponent of any hemoglobin present in the sample and bound in theglobin-antiglobin complex becomes bound to the immobilized antiglobincapture antibody where it is detectable.

In this dual test aspect, the present invention may be used to diagnosegastrointestinal tract bleeding by analysing fecal samples for thepresence of blood. Without limiting the present invention to any onetheory or mode of action, the chromogen test will positively identifybleeding from any part of the gastrointestinal tract (that is, both theupper and lower regions of the tract) since it detects the hemecomponent of hemoglobin and heme is relatively resistant to breakdown inthe small intestine (the upper gastrointestinal tract). The globincomponent of hemoglobin however, does not survive passage through theupper gastrointestinal tract. A positive globin result in a fecal sampletherefore indicates that bleeding has occurred in the lowergastrointestinal tract. Accordingly, by applying a combined dualimmunological and non-immunological based test, it is possible todifferentiate between upper and lower gastrointestinal tract bleedingwherein a positive heme result together with a negative globin resultindicates upper gastrointestinal tract bleeding, and a positive hemeresult together with a positive globin result indicates lowergastrointestinal tract bleeding. This is of particular importance, forexample to the diagnosis of colorectal cancer, the symptoms of whichinclude lower gastrointestinal tract bleeding.

Further features of the test device and method of the present inventionare more fully described below with reference to the accompanyingdrawing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a “dual test” device inaccordance with a preferred embodiment of the present invention.

FIGS. 2A and 2B are schematic representations of an alternative devicein accordance with the present invention. FIG. 2A shows a FOBT teststrip, and FIG. 2B shows a FOBT test strip in housing.

FIG. 3 is a schematic representation of a further alternative device inaccordance with the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows schematically a dual test strip format in accordance withthe present invention, which comprises the following components inliquid-conductive communication in a single test strip, as follows:

-   -   TMB impregnated indicator paper (1);    -   Substrate (e.g. dried cumene peroxide) impregnated paper (2);    -   Sample pad (e.g. non woven polyester impregnated detergent, e.g.        with Triton X100) (3);    -   Conjugate pad (e.g. gold labeled anti-human globin antibodies)        (4);    -   Solid phase (e.g. nitrocellulose membrane with immobilized        anti-human globin antibody line and a procedural control line)        (5);    -   Absorbent pad (6).

Components 1, 2 and 3 constitute the basic components of the test deviceof the present invention. If desired, components 1 and 2 may becombined, provided precautions are observed to provide storage stabilityof the various reagents. Components 4, 5 and 6 constitute the additionalcomponents of the preferred embodiment of this invention which includesan immunochemical test strip for detecting human globin as an indicatorof lower intestinal bleeding.

Sample, for example, a stool sample from a digital rectal examination(DRE), or a water sample taken from around a stool in a toilet bowl, isapplied to the sample pad 3, where any red blood cells are lysed by thedetergent impregnated in the sample pad, and developer solution isadded. The developer solution may be water, or may include buffer,ethanol and other reagents that assist the reactions and that arecompatible with both types of test. From the sample pad, the developersolution mobilizes any hemoglobin released from red blood cells andmoves laterally from pad 3 through the flanking regions of the teststrip in both directions.

Heme, if present, mixes with the pseudoperoxidase substrate in substrateregion 2 and then mixes with the chromogenic indicator in indicatorregion 1, where the color accumulates at the end of region 1.

Globin, if present, is detected in the detection region 5 after labelingwith the gold-labeled antibody conjugate in substrate region 4. Excessdeveloper solution and other reagent accumulate in the absorbent pad 6.

Clearly, the dual test strip illustrated in FIG. 1 may be encased in ahousing adapted for receipt of the sample on pad 3, with provision (e.g.windows or similar apertures) for visual, or instrumented, detection ofthe results in indicator region 1 and detection region 5.

Example 1

Reagent solutions were prepared based on Lam, U.S. Pat. No. 4,071,318,as follows:

Solution A:

Water 10 mL Trisodium citrate 213 mg Citric acid 147 mg EDTA 6.7 mgSodium lauryl sulfate 67 mg Methyl sulfone 667 mg Acetone 1.67 mL

Solution B (Indicator):

Tetra methyl benzidine (TMB) 26.7 mg dissolved in Dimethylsulfoxide 1.67mL

Solution C (Substrate):

Cumene hydroperoxide 133.3 mg 6-methoxy quinoline 33.3 mgTriethanolamine borate 667 mg Solution A 5 mL

To prepare reactive paper, solutions A, B and C were mixed just beforeuse and added to Whatmans #1 paper until the paper was soaked. Theimpregnated reactive paper was hung vertically to drain excess liquidand dried in a warm air current for approximately 30 minutes. Theactivity of the reactive paper was confirmed by diluting blood obtainedfrom a finger prick in water and adding the dilutions to small pieces ofthe reactive paper. A 1/100 dilution gave an instant strong blue-greencolor, 1/1000 produced a strong blue color, 1/10,000 produced a slowerdeveloping green color, whereas the 1/100,000 dilution produced aborderline pale blue after 1-2 minutes. Water alone added to thereactive paper produced no color, even when left until dry.

The reactive paper was tested in a device constructed as shown in FIGS.2A (FOBT test strip) and 2B (FOB test strip in housing). FOBT teststrips were prepared by laminating the pads A, B, C and D as shown inFIG. 2A with double sided adhesive (3M #465, 3M MN) to a white plastic(high impact polystyrene) backing (D) and cutting the laminate into teststrips approximately 10 mm wide. The test strips were then placed in awaterproof cardboard housing (G) with a port (E) for sample and reagentaddition and a window (F). For ease of reading the test result, theinterface between pads B and C was located centrally in the observationwindow (F) of the test housing (G).

In the test strips shown in FIG. 2A:

Pad A: Non-woven polyester fabric (e.g. Ahlstrom 6613, Ahlstrom, Pa.)impregnated with 0.1% Triton X-100 detergent.

Pad B: Reactive paper.

Pad C: White plastic barrier tape.

Pad D: Backing.

In this test strip, pad A is the sample application pad, and pad B is acombined substrate/indicator region.

Blood diluted 1/1000 in water was applied to pad A of the test strip ofFIG. 2A via the sample port (E) followed by three drops of water. Theliquid migrated from pad A via pad B so that within 25 seconds a strongblue color accumulated at the end of pad B against the white impermeablebarrier of pad C. Water alone added to Pad A produced no color in thetest window.

Example 2

For long term stability of the FOBT test strip, the substrate andindicator regions were prepared and laminated separately in a test stripconstructed as shown in FIG. 3 as a modification of the test strip shownin FIG. 2A.

In the test strip of FIG. 3, pad A is the sample application pad, pad B1is the substrate region and pad B2 is the indicator region. The teststrip is prepared as described in Example 1, using substrate paper B1and indicator paper B2 instead of reactive paper B, as follows:

-   -   Substrate paper B1: Solution C was prepared, soaked into        Whatman's #1 filter paper and drip-dried.    -   Indicator paper B2: Solutions A and B were mixed, soaked into        Whatman's #1 filter paper and drip-dried.    -   Both papers B1 and B2 were then fully dried in forced air at low        heat.        10 μL of human blood diluted 1/1000 in water was added to pad A        of the test strip of FIG. 3 followed by three drops of a reagent        comprised of Bovine serum albumin (3%), Ethanol (10%) and sodium        azide in 40 mM sodium borate buffer, pH 8.5. Blue color        accumulated at the interface of membrane B2 and C. No color        developed with a water sample alone.

In an alternative embodiment, the substrate Solution C may beincorporated into pad A during manufacture, and pad B1 omitted from thetest strip.

Example 3

FOBT test strips, as described for Example 1, were used in combinationwith commercially available immunochemical (ICT) test strips (InSureFIT, Enterix Inc., NJ). These ICT test strips are used for detection ofhuman globin as an indicator of lower intestinal bleeding.

The two test strips (FOBT and ICT) were laid end to cnd so that theorigin of the ICT strip was in contact with pad A of the test strip asdescribed in Example 1. 10 μL of a 1/1000 dilution of human blood inwater was added to pad A, followed by four drops of the reagentdescribed in Example 2. The sample migrated in both directions from thepoint of application and both tests developed a positive result. Whenwater containing diluted blood taken from a beef sample was tested inthe same manner it gave a positive result with the FOBT test strip(i.e., positive for hemoglobin), but a negative result with the ICT test(i.e., negative for human globulin). Water alone gave a negative resultwith both tests.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. A method for the detection of hemoglobin in a biological sample,comprising the steps of: (i) providing a carrier matrix which comprisesa sample application region, a first and a second substrate region, anda first and a second indicator region, wherein a biological sample hasbeen applied to the sample application region; (ii) contacting the firstsubstrate region with the biological sample, wherein the first substrateregion in liquid-conductive communication with, or combined with, thesample application region, wherein a pseudoperoxidase substratecomprising a peroxidase or hydroperoxidase reagent is contacted with thesample; and wherein the pseudoperoxidase substrate is present in thefirst substrate region in a dried state prior to contacting with thebiological sample; (iii) contacting the sample and pseudoperoxidasesubstrate with the first indicator region, wherein the first indicatorregion in liquid-conductive communication with, or combined with, thefirst substrate region and having an indicator applied thereto orimpregnated therein, wherein the sample and substrate are contacted withthe indicator which produces a detectable response in the presence ofheme and the pseudoperoxidase substrate; (iv) contacting a secondsubstrate region with the biological sample, wherein the secondsubstrate region is in liquid-conductive communication with the sampleapplication region and having a detectable anti-globin immunointeractivemolecule applied thereto or impregnated therein, wherein the sample iscontacted with a detectable anti-globin immunointeractive molecule toform a detectable globin-antiglobin complex in the presence of globin;and (v) contacting the detectable globin-antiglobin complex with asecond indicator region, wherein the second indicator region is inliquid-conductive communication with the second substrate region andhaving an anti-globin immunointeractive molecule immobilized therein,wherein the detectable globin-antiglobin complex is contacted with animmobilized anti-globin immunointeractive molecule to immobilize thedetectable globin-antiglobin complex.
 2. The method of claim 1, whereinthe sample application region and the first substrate region arecombined in a combined sample application/substrate region of thecarrier matrix, wherein the biological sample is contacted with thepseudoperoxidase substrate in the combined sample application/substrateregion of the carrier matrix.
 3. The method of claim 1, wherein thesample application region and the first substrate region are separateregions of the carrier matrix which are in liquid-conductivecommunication, and wherein the biological sample is contacted with thepseudoperoxidase substrate in separate regions of the carrier matrixwhich are in liquid-conductive communication with the sample applicationregion.
 4. The method of claim 3, wherein the biological sample iscontacted with the substrate in the first substrate region by permittingor causing the sample to flow from the sample application region to thefirst substrate region of the carrier matrix.
 5. The method of claim 1,wherein the biological sample is contacted with the pseudoperoxidasesubstrate and indicator in a combined first substrate/first indicatorregion of the carrier matrix.
 6. The method of claim 1, wherein thefirst substrate region and the first indicator region are separateregions of the carrier matrix which are in liquid-conductivecommunication, wherein the biological sample and the substrate arecontacted with the indicator in the separate first indicator region ofthe carrier matrix which is in liquid-conductive communication with thefirst substrate region.
 7. The method of claim 6, wherein the biologicalsample and the substrate are contacted with the indicator in the firstindicator region by permitting or causing the sample and substrate toflow from the first substrate region to the first indicator region ofthe carrier matrix.
 8. The method of claim 1, wherein the secondsubstrate region is in liquid conductive communication with the sampleapplication region, and the biological sample is permitted or caused toflow from the sample application region to the second substrate region.9. The method of claim 1, wherein the second indicator region is inliquid-conductive communication with the second substrate region, andthe detectable globin anti-globin complex is permitted or caused to flowfrom the second substrate region to the indicator region.
 10. The methodof claim 1, wherein the biological sample is a sample derived from thegastrointestinal tract of a patient.
 11. The method of claim 10, whereinthe sample comprises feces.
 12. The method of claim 10, wherein thepatient is a human.
 13. A method for distinguishing uppergastrointestinal tract bleeding from lower gastrointestinal tractbleeding, comprising the steps of: (i) providing a carrier matrix whichcomprises a sample application region, a first and a second substrateregion, and a first and a second indicator region, wherein a biologicalsample has been applied to the sample application region; (ii)contacting the first substrate region with the biological sample,wherein the first substrate region in liquid-conductive communicationwith, or combined with, the sample application region, wherein apseudoperoxidase substrate comprising a peroxidase or hydroperoxidasereagent is contacted with the sample; and wherein the pseudoperoxidasesubstrate is present in the first substrate region in a dried stateprior to contacting with the biological sample; (iii) contacting thesample and pseudoperoxidase substrate with the first indicator region,wherein the first indicator region is in liquid-conductive communicationwith, or combined with, the first substrate region and having anindicator applied thereto or impregnated therein, wherein the sample andsubstrate are contacted with the indicator which produces a detectableresponse in the presence of heme and the pseudoperoxidase substrate;(iv) contacting a second substrate region with the biological sample,wherein the second substrate region is in liquid-conductivecommunication with the sample application region and having a detectableanti-globin immunointeractive molecule applied thereto or impregnatedtherein, wherein the sample is contacted with a detectable anti-globinimmunointeractive molecule to form a detectable globin-antiglobincomplex in the presence of globin; (v) contacting the detectableglobin-antiglobin complex with a second indicator region, wherein thesecond indicator region is in liquid-conductive communication with thesecond substrate region and having an anti-globin immunointcractivemolecule immobilized therein, wherein the detectable globin-antiglobincomplex is contacted with an immobilized anti-globin immunointeractivemolecule to immobilize the detectable globin-antiglobin complex; whereindetecting heme alone in the biological sample is indicative of uppergastrointestinal tract bleeding and detecting of heme and globin in thebiological sample is indicative of lower gastrointestinal tractbleeding.
 14. The method of claim 13, wherein the sample applicationregion and the first substrate region are combined in a combined sampleapplication/substrate region of the carrier matrix, wherein thebiological sample is contacted with the pseudoperoxidase substrate inthe combined sample application/substrate region of the carrier matrix.15. The method of claim 13, wherein the sample application region andthe first substrate region are separate regions of the carrier matrixwhich are in liquid-conductive communication, and wherein the biologicalsample is contacted with the pseudoperoxidase substrate in separateregions of the carrier matrix which are in liquid-conductivecommunication with the sample application region.
 16. The method ofclaim 15, wherein the biological sample is contacted with the substratein the first substrate region by permitting or causing the sample toflow from the sample application region to the first substrate region ofthe carrier matrix.
 17. The method of claim 13, wherein the biologicalsample is contacted with the pseudoperoxidase substrate and indicator ina combined first substrate/first indicator region of the carrier matrix.18. The method of claim 13, wherein the first substrate region and thefirst indicator region are separate regions of the carrier matrix whichare in liquid-conductive communication, wherein the biological sampleand the substrate are contacted with the indicator in the separate firstindicator region of the carrier matrix which is in liquid-conductivecommunication with the first substrate region.
 19. The method of claim18, wherein the biological sample and the substrate are contacted withthe indicator in the first indicator region by permitting or causing thesample and substrate to flow from the first substrate region to thefirst indicator region of the carrier matrix.
 20. The method of claim13, wherein the second substrate region is in liquid conductivecommunication with the sample application region, and the biologicalsample is permitted or caused to flow from the sample application regionto the second substrate region.
 21. The method of claim 13, wherein thesecond indicator region is in liquid-conductive communication with thesecond substrate region, and the detectable globin anti-globin complexis permitted or caused to flow from the second substrate region to theindicator region.
 22. The method of claim 13, wherein the biologicalsample is a sample derived from the gastrointestinal tract of a patient.23. The method of claim 22, wherein the sample comprises feces.
 24. Themethod of claim 22, wherein the patient is a human.